Beta receptor density in human lymphocyte membranes: changing with aging?

The number of beta adrenergic binding sites (Bmax) in human lymphocyte membranes has been reported to decrease, remain the same, or increase with age. In order to address this issue, we used two highly specific beta receptor ligands with lymphocytes from healthy aged (range: 51-90 years) and young (19-39 years) subjects in two separate studies. Because depression can reduce Bmax, potential aged subjects were excluded if they had high scores on tests for depression. Bmax was higher in the aged group of each study (33% higher in the first, p less than .01, and 72.5% in the second, p less than .02); the results were similar in both studies. Antagonist affinity did not differ between young and aged groups in either study. We suggest that some of the discrepancies in the literature could be due to differences in age ranges used or to inclusion of depressed subjects in prior studies.

Inhibition of alpha-ketoglutarate dehydrogenase activity by a distinct population of autoantibodies recognizing dihydrolipoamide succinyltransferase in primary biliary cirrhosis.

Sera from patients with primary biliary cirrhosis contain autoantibodies that recognize mitochondrial proteins. Five of the target autoantigens have now been identified as enzymes of three related multienzyme complexes: the pyruvate dehydrogenase complex, the branched chain alpha-ketoacid dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex. Each complex consists of component enzymes designated E1, E2 and E3. In this report, we confirm that primary biliary cirrhosis sera react with dihydrolipoamide succinyltransferase, the E2 component of alpha-ketoglutarate dehydrogenase complex. Seventy-three of 188 (39%) primary biliary cirrhosis sera reacted with alpha-ketoglutarate dehydrogenase complex-E2 when immunoblotted against purified alpha-ketoglutarate dehydrogenase complex; one of these sera also reacted with the E1 component. In addition, primary biliary cirrhosis sera possessing alpha-ketoglutarate dehydrogenase complex-E2 reactivity specifically inhibited enzyme function of alpha-ketoglutarate dehydrogenase complex. Enzyme activity was not affected by primary biliary cirrhosis sera that contained autoantibodies to pyruvate dehydrogenase complex-E2 and/or branched chain alpha-ketoacid dehydrogenase complex-E2, which lacked alpha-ketoglutarate dehydrogenase complex-E2 reactivity. Furthermore, affinity-purified primary biliary cirrhosis sera against alpha-ketoglutarate dehydrogenase complex-E2 inhibited only alpha-ketoglutarate dehydrogenase complex activity but did not alter enzyme activity of either pyruvate dehydrogenase complex or branched chain alpha-ketoacid dehydrogenase complex. Finally, alpha-ketoglutarate dehydrogenase complex-E2 specific affinity-purified antisera did not react on immunoblot with any component enzymes of pyruvate dehydrogenase complex or branched chain alpha-ketoacid dehydrogenase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary biliary cirrhosis. Inhibition of pyruvate dehydrogenase complex activity by autoantibodies specific for E1 alpha, a non-lipoic acid containing mitochondrial enzyme.

Antimitochondrial antibodies (AMA) are serologically characteristic of patients with PBC. Four Ag recognized by AMA have been recently identified, including protein X and the acyltransferases of three related multienzyme complexes: the pyruvate dehydrogenase complex (PDC), the branched-chain alpha-ketoacid dehydrogenase complex, and the alpha-ketoglutarate dehydrogenase complex. Each of these enzymes contains one or more lipoyl moieties as part of a major functional site. In addition, epitope mapping has suggested that the AMA target is this lipoic acid-binding region. In this report we demonstrate that sera from patients with PBC also recognize the E1 component (pyruvate dehydrogenase, EC 1.2.4.1) of PDC. PDC-E1 is composed of alpha and beta-chains. Reactivity with the 41 kD alpha chain was detected in 80 of 120 (66%) PBC sera by immunoblotting against purified PDC-E1; 2 of 120 sera also demonstrated reactivity with the 34 kD beta chain. In contrast, sera from patients with SLE, chronic active hepatitis, or progressive sclerosing cholangitis as well as sera from healthy volunteers did not react with PDC-E1. Furthermore, affinity-purified PBC sera against PDC-E1 alpha were able to inhibit PDC enzyme activity, whereas control sera could not. PDC-E1 alpha is now the fifth mitochondrial autoantigen of PBC to be identified. Similar to the four previously identified autoantigens, AMA appear to be directed to a functional site of PDC-E1 alpha inasmuch as they are able to inhibit enzyme function. However, PDC-E1 alpha is also unique in that it is the first identified mitochondrial autoantigen which does not contain lipoic acid.

Antimitochondrial antibodies of primary biliary cirrhosis recognize dihydrolipoamide acyltransferase and inhibit enzyme function of the branched chain alpha-ketoacid dehydrogenase complex.

Antimitochondrial antibodies (AMA) recognizing the acetyltransferase (E2) of the pyruvate dehydrogenase (PDH) complex have been previously well-documented and the immunodominant epitope mapped. In this study, we demonstrate that sera from patients with primary biliary cirrhosis (PBC) react with another lipoic acid containing acyltransferase enzyme, namely the E2 of the branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Indeed, 85/120 (71%) sera from patients with PBC reacted with BCKD-E2 by immunoblotting against purified BCKD complex. In contrast, sera from patients with chronic active hepatitis or progressive sclerosing cholangitis as well as sera from healthy volunteers did not react with any component enzymes of the BCKD complex. More importantly, BCKD enzyme activity was inhibited after incubation of the BCKD complex with either PBC sera against BCKD-E2 or with affinity purified antisera to BCKD-E2. Enzyme activity was unaltered by control sera or with PBC sera that reacted with PDH-E2 but not BCKD-E2. Furthermore, immunoblots of purified mitochondria probed with PBC sera absorbed with BCKD-E2 demonstrated that BCKD-E2 and PDH-E2 are each recognized by distinct AMA populations which do not cross-react. In addition, affinity purified PBC sera against BCKD-E2 did not react with PDH-E2 nor inhibit PDH enzyme activity, thus providing further evidence that BCKD-E2 and PDH-E2 are recognized by separate AMA. These data further suggest that the BCKD-E2 epitope recognized by AMA contains, or is close to, a functional domain of this enzyme. The availability of cDNA clones encoding BCKD-E2 and PDH-E2 will allow the study of how key metabolic enzymes may be involved in the immunology and pathology of PBC.

Autoantibodies to mitochondria in systemic sclerosis. Frequency and characterization using recombinant cloned autoantigen.

Mitochondrial autoantibodies, a hallmark of primary biliary cirrhosis (PBC), have been widely described for many years in patients with systemic sclerosis, and there have been several reports of the concurrence of systemic sclerosis and PBC. However, there is very little information with respect to the significance of these autoantibodies or any definitive evidence that the antigens involved represent the mitochondrial autoantigens (M2 complex) described in PBC. We have cloned and sequenced a rat complementary DNA which encodes for all the epitopes recognized by autoantibodies to the major, or 70-kd, mitochondrial autoantigen in patients with PBC. Using this recombinant fused autoantigen, as well as by immunoblotting with human placental mitochondria, we tested for antimitochondrial antibody specificity in sera from 250 patients with systemic sclerosis. Nineteen sera (7.6%), including those from patients with CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) and diffuse scleroderma, had reactivity with human placental mitochondria proteins by immunoblot testing. All 19 sera reacted with the M2 complex. All sera that reacted with the 70-kd protein likewise reacted with the recombinant cloned autoantigen. The predominant autoantibody isotype to the 70-kd protein was IgG3. Interestingly, the 70-kd protein is 11% proline, an amino acid which is frequently preceded by hydrophobic amino acids.

Influence of dietary fatty acid concentration and geometric configuration on murine mammary tumorigenesis and experimental metastasis.

To investigate the influence of fatty acid geometric isomers on the growth and experimental metastasis of mammary tumors, mice were fed diets containing fat high in either cis or trans fatty acids. The cis fat was prepared to have a fatty acid composition similar to that of the trans fat; both were provided at 5 or 20% (by weight) of the diet. Line 168 mammary tumor cells were transplanted: 1) subcutaneously into female BALB/c mice to observe the effects of dietary fat on latency and local tumor growth, and 2) intravenously to observe influences on experimental metastasis. No differences in the latency or rate of primary tumor growth were observed among the groups fed the diets containing cis or trans fatty acids. In addition, there were no differences in fatty acid composition except the levels of trans-C18:1 in the primary tumor cells among the groups fed the experimental diets. Livers and spleens from animals fed both the 5 and 20% cis diet contained significantly more viable radiolabeled tumor cells than those fed the trans diets. Although body weight and composition were not significantly different among the groups, livers from animals fed the diets containing trans fatty acids were significantly heavier than those fed diets containing only cis fatty acids. Thus, trans fatty acids behaved similarly to cis fatty acids with respect to promotion of transplantable mammary tumor growth but trans fatty acids were less effective than cis fatty acids in promoting the blood-borne implantation and distant survival of the tumor cells.